Composition and method for preserving progenitor cells

ABSTRACT

The invention relates to a protein material which is effective to preserve progenitor cells, such as hematopoietic progenitor cells. The protein has an amino acid sequence comprising AQSLSFSFTKFD (SEQ ID NO:1) and a molecular weight of about 12-20 kD, or has an amino acid sequence comprising VVAVEFD (SEQ ID NO:3) and a molecular weight of about 15-20 kD. Heterodimers of the protein are described, and multimers thereof. Methods of using the protein of the invention for preserving progenitor cells in vitro, ex vivo, and in vivo are also described. The invention, therefore, include methods such as myeloablation therapies for cancer treatment wherein myeloid reconstitution is facilitated by means of the specified protein. Other therapeutic utilities are also enabled through the invention, for example, expanding progenitor cell populations ex vivo to increase chances of engraftation, improving conditions for transporting and storing progenitor cells, and facilitated gene therapy to treat and cure a broad range of life-threatening hematologic diseases.

This is a continuation of application Ser. No. 08/825,369, filed on Mar.28, 1997, now U.S. Pat. No. 6,084,060, which was a continuation-in-partof application Ser. No. 08/762,537, filed on Dec. 9, 1996, nowabandoned.

BACKGROUND OF THE INVENTION

The invention relates to an agent and method for use in connection withprogenitor cells. More specifically, the invention relates to a proteincapable of preserving progenitor cells and a method of using the proteinfor maintaining and preserving progenitor cells.

Each day the bone marrow generates and releases into the circulationseveral billion fully-differentiated, functional blood cells. Productionof these cells derives from a small stock of quiescent progenitor cells(including the most primitive stem cells and other less primitive butstill immature progenitors) by a process called hematopoiesis (Zipori1992). The most primitive stem cells have the capacity to generate>10¹³cells containing all blood lineages (Turhan et al. 1989). The productionof such a large number of cells is achieved by extensive proliferationcoupled with successive differentiation steps leading to a balancedproduction of mature cells. Progenitor cells progressively lose theircapacity to generate multiple cells lineages and eventually producecells of one or two cell lineages.

Soluble regulators and cell-cell interactions mediate differentiationdirections of immature progenitors through a tightly-controlled butinadequately understood process. Several of the body's soluble factorshave been isolated and characterized both in culture and in animals(see, e.g., Ogawa (1993) and references therein). Regulators such as thecolony stimulating factors (e.g., IL3, GM-CSF, G-CSF, M-CSF) not onlyinduce proliferation and differentiation of progenitors capable ofproducing cells of either multiple cell lineages (IL3 and GM-CSF) orsingle cell lineages (G-CSF and M-CSF), but also maintain viability oftheir respective progenitors. Other regulators such as interleukin-1(IL1), the kit ligand (KL), and thrombopoietin (Borge et al. 1996)increase viability of multipotential progenitors in addition to otherfunctions. No known cytokines alone or in combination can maintainviability of primitive progenitors in liquid culture without stromalsupport beyond a few days.

Regulation of primitive stem cells appears to differ from that ofimmature, multilineage progenitors. Stem cells are primarily quiescentand do not appear to respond immediately to regulators that induceproliferation and differentiation. Maintenance of these cells in thebody is mediated via cell-cell interactions and soluble regulators.Maintenance of quiescent stem cells in vitro has been achieved byculturing cells on adherent stromal layers with soluble regulators suchas IL3, IL6, KL and LIF (Young et al. 1996). Recently, the addition ofFL to this complex culture has been found to extend maintenance ofquiescent stem cells from a few weeks to three months (Shah et al.1996). Establishing stromal cells cultures is not easily applicable toclinical settings.

Lectins, defined as carbohydrate-binding proteins other than antibodiesor enzymes, (Baronedes 1988), are widespread among plants, prokaryotes,and eukaryotes. Each lectin recognizes a specific carbohydrate moiety,and forms a non-covalent bond with the carbohydrate through astereochemical fit of complementary molecules (e.g., hydrophobicpocket). Carbohydrates are widely present on cell surfaces (in the formsof glycoproteins, glycolipids, and polysaccharides), and appear tomediate cell-cell contacts including cell recognition (Sharon et al.1989). Abnormal glycosylation patterns are associated with disease bycausing alterations in a protein's conformation, stability, or proteaseresistance (Dwek 1995).

Gowda et al. (1994) describe the isolation of a mannose-glucose-specificlectin from the hyacinth bean (Dolichos lab lab). Purification andsequencing of this lectin is said to indicate that the protein includestwo nonidentical subunits. The Gowda et al. publication describesevolutionary relationships of the lectin to other lectins, but does notascribe any function to the protein beyond saccharide-binding in theplant source.

Cell agglutinating properties of certain plant lectins have been knownfor over 100 years. Certain lectins have been used as tools inimmunology laboratories as potent, specific activators of T lymphocytes(phytohemagglutinin (PHA) and concanavalin A (ConA)) and B lymphocytes(pokeweed mitogen (PWM)) for over 30 years (Sharon et al. 1989). Somelectins have also been used to isolate hematopoietic progenitors forover 15 years (Gabius 1994a). Large numbers of cancer patients in Europehave received mistletoe lectin (Viscum album) intravenously as acandidate cancer therapy without major complications (Gabius 1994b).Whether these plant lectins act on mammalian cells via de novo means, orsimply mimic their functional mammalian homologs is not yet known. Nolectin has yet been successfully developed as a human therapeutic.

In view of the above considerations, it is clear that regulation of thehematopoietic process remains incompletely understood. Most solubleregulators identified, such as the colony stimulating factors andinterleukins, induce proliferation and differentiation of progenitorscells in culture and their levels in the blood circulation increaseduring times of hematopoietic stress (e.g., blood loss, infection). Forexample, U.S. Pat. No. 4,808,611 describes a method of using IL1 and acolony stimulating factor to induce proliferation and differentiation ofhemopoietic stem cells. Some soluble regulators, such as IL1, IL6, KL,FL, and Tpo, appear to provide increase viability of stem cells withoutdirectly affecting proliferation and differentiation. But no knownsoluble regulators, alone or in combination, have yet been reported thatenable maintenance and amplification of stem cells populations in vitrowithout stromal cells. As a consequence, numerous potential therapeuticapproaches to diseases such as cancer and genetic blood diseases remainunavailable.

Accordingly, it is one of the purposes of this invention to overcome theabove limitations in methods of regulating hematopoietic processes, byproviding a factor and method of protecting, maintaining, and expandinghematopoietic progenitor cell populations. It is another purpose of theinvention to provide means for protecting the integrity of thehematopoietic processes in vivo as an adjunct to therapeutic treatmentsrelated to cancer and other diseases which can otherwise adverselyimpact upon the hematopoietic system.

SUMMARY OF THE INVENTION

It has now been discovered that these and other objectives can beachieved by the present invention, which provides a protein whichpreserves progenitor cells and a method of using the protein. Theprotein has an amino acid sequence comprising AQSLSFSFTKFD (SEQ ID NO:1)and a molecular weight of about 12-20 kD, or has an amino acid sequencecomprising VVAVEFD (SEQ ID NO:3) and a molecular weight of about 15-20kD. The method of preserving progenitor cells comprises contactingprogenitor cells with a protein, having an amino acid sequencecomprising AQSLSFSFTKFD (SEQ ID NO:1) and a molecular weight of about12-20 kD, or having an amino acid sequence comprising VVAVEFD (SEQ IDNO:3) and a molecular weight of about 15-20 kD, in an amount sufficientto preserve the progenitor cells.

In one embodiment, the invention includes a method of treating a mammalin need of hematopoietic therapy. Here the method comprises:

a) obtaining a tissue sample from the mammal, the tissue samplecomprising hematopoietic progenitor cells;

b) culturing the progenitor cells in the presence of a protein whichpreserves the progenitor cells, to provide cultured cells enriched inthe progenitor cells, wherein the protein has an amino acid sequencecomprising AQSLSFSFTKFD (SEQ ID NO:1) and a molecular weight of about12-20 kD, or has an amino acid sequence comprising VVAVEFD (SEQ ID NO:3)and a molecular weight of about 15-20 kD;

c) subjecting the mammal to conditions sufficient to effectmyeloablation; and

d) administering the cultured cells to the mammal following themyeloablation to reconstitute the hematopoietic system of the mammal.

In an alternative embodiment, the invention includes a method ofenriching progenitor cells. The method comprises culturing progenitorcells in a progenitor-preserving amount of a protein having an aminoacid sequence comprising VVAVEFD (SEQ ID NO:3) and a molecular weight ofabout 15-20 kD, or having an amino acid sequence comprising AQSLSFSFTKFD(SEQ ID NO:1) and a molecular weight of about 12-20 kD, wherein theprotein specifically preserves the progenitor cells, and wherein theculturing is performed under conditions permitting preservation ofprogenitor cells while permitting the number of differentiated cells todecrease.

In still another embodiment, the invention includes a method ofimproving hematopoietic competence in a mammal. In this case, the methodcomprises:

a) culturing a tissue sample comprising hematopoietic progenitor cellsin a growth medium containing a protein having an amino acid sequencecomprising AQSLSFSFTKFD (SEQ ID NO:1) and a molecular weight of about12-20 kD, or having an amino acid sequence comprising VVAVEFD (SEQ IDNO:3) and a molecular weight of about 15-20 kD, in an amount sufficientto preserve the progenitor cells and to provide cultured cells enrichedin the progenitor cells; and

b) transfusing the enriched cultured cells to the mammal to provideprogenitor cells for generating blood cellular components in the mammal.

In yet another embodiment, the invention further includes a method oftransfecting an exogenous DNA sequence into somatic cells, whichincludes the improvement comprising transfecting progenitor cellsselectively preserved by a protein having an amino acid sequencecomprising AQSLSFSFTKFD (SEQ ID NO:1) and a molecular weight of about12-20 kD, or having an amino acid sequence comprising VVAVEFD (SEQ IDNO:3) and a molecular weight of about 15-20 kD.

In a further embodiment, the invention includes a composition formaintaining viability of progenitor cells ex vivo, comprising a cellgrowth medium and a protein which preserves progenitor cells, whereinthe protein has an amino acid sequence comprising AQSLSFSFTKFD (SEQ IDNO:1) and a molecular weight of about 12-20 kD, or has an amino acidsequence comprising VVAVEFD (SEQ ID NO:3) and a molecular weight ofabout 15-20 kD.

Moreover, in yet another embodiment, the invention includes, a methodfor preserving progenitor cells in a mammal. The method of thisembodiment comprises:

a) administering to the mammal a protein which specifically preservesprogenitor cells, the protein having an amino acid sequence comprisingAQSLSFSFTKFD (SEQ ID NO:1) and a molecular weight of about 12-20 kD, orhaving an amino acid sequence comprising VVAVEFD (SEQ ID NO:3) and amolecular weight of about 15-20 kD, in an amount sufficient to preserveprogenitor cells of the mammal in a substantially non-proliferativestate;

b) exposing the mammal to myeloablative conditions sufficient to effectablation of proliferating myeloid cells but sparing non-proliferatingprogenitor cells; and

c) following the exposing, reestablishing proliferation ordifferentiation of the preserved progenitor cells.

These and other advantages of the present invention will be appreciatedfrom the detailed description and examples which are set forth herein.The detailed description and examples enhance the understanding of theinvention, but are not intended to limit the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention have been chosen for purposes ofillustration and description, but are not intended in any way torestrict the scope of the invention. The preferred embodiments ofcertain aspects of the invention are shown in the accompanying drawings,wherein:

FIG. 1 shows an electrophoretic separation of protein illustrating thealpha and beta subunit structure of the pylartin protein obtained fromhyacinth beans.

FIG. 2A is a graph of the effect of the protein of the invention onflk2-transfected 3T3 cells in the presence of IL1;

FIG. 2B is a graph of the effect of the protein of the invention onflk2-transfected 3T3 cells in the absence of IL1;

FIG. 2C is a graph of the effect of the protein of the invention onuntransfected 3T3 cells in the presence of IL1;

FIG. 2D is a graph of the effect of the protein of the invention onuntransfected 3T3 cells in the absence of IL1.

FIG. 3 is a series of histograms comparing the protein of the invention,obtained from red kidney beans, and from hyacinth beans, against culturemedium control, showing that the protein of the invention specificallypreserves murine CD34⁺ cells in liquid culture, with and without acocktail of cytokines including mIL1, mIL3, and mKL.

FIG. 4 is a series of histograms comparing the protein of the invention,obtained from red kidney beans or hyacinth beans, against culture mediumcontrol, showing that the protein of the invention specificallypreserves murine progenitor cells in a methylcellulose colony assay,with and without a cocktail of cytokines including mIL1, mIL3, and mKL.

FIG. 5 is a series of histograms showing the results of flow cytometricstudy of six different cell surface markers showing that the protein ofthe invention specifically preserves primitive murine progenitors.

FIG. 6A is a set of histograms comparing the protein of the inventionagainst culture medium control, showing that the protein of theinvention specifically preserves CD34⁺ human cord blood cells in liquidculture for six days;

FIG. 6B is a complementary set of histograms showing that the protein ofthe invention specifically preserves CD34⁺ human cord blood cells inliquid culture for 17 days.

FIG. 7 is a set of two histograms comparing the protein of the inventionagainst a control, illustrating that the pylartin protein, either aloneor in combination with IL1-a or FL preserves or increases the number ofhuman progenitor cells in a methylcellulose colony assay.

FIGS. 8A-8C are a set of histograms showing that the protein of theinvention, and not FL, preserves primitive hematopoietic progenitorcells in liquid culture after 15 and 21 days.

FIGS. 9A and 9B are a set of histograms showing that the protein of theinvention preserves hematopoietic progenitors in a dose-responsivemanner.

FIG. 10 is a pair of histograms, comparing the protein of the inventionagainst medium control, illustrating that the pylartin protein preservesprogenitors by preventing IL3-induced differentiation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to a protein which preservesprogenitor cells, and a method for use of the protein in preserving andmaintaining progenitor cells. By “protein” is meant any isolated naturalor synthetic oligo- or polypeptide comprising a sequence of amino acidresidues linked by peptide bonds.

As used herein, “progenitor cell” refers to any somatic cell which hasthe capacity to generate fully differentiated, functional progeny bydifferentiation and proliferation. Progenitor cells include progenitorsfrom any tissue or organ system, including, but not limited to, blood,nerve, muscle, skin, gut, bone, kidney, liver, pancreas, thymus, and thelike. Progenitor cells are distinguished from “differentiated cells,”which are defined as those cells which may or may not have the capacityto proliferate, i.e., self-replicate, but which are unable to undergofurther differentiation to a different cell type under normalphysiological conditions. Moreover, progenitor cells are furtherdistinguished from abnormal cells such as cancer cells, especiallyleukemia cells, which proliferate (self-replicate) but which generallydo not further differentiate, despite appearing to be immature orundifferentiated.

Progenitor cells include all the cells in a lineage of differentiationand proliferation prior to the most differentiated or the fully maturecell. Thus, for example, progenitors include the skin progenitor in themature individual, which is capable of differentiation to only one typeof cell, but which is itself not fully mature or fully differentiated.Production of mature, functional blood cells results from proliferationand differentiation of “unipotential progenitors,” i.e., thoseprogenitors which have the capacity to make only one type of one type ofblood cell. For red blood cell production, a progenitor called a “CFU-E”(colony forming unit-erythroid) has the capacity to generate two to 32progeny cells.

Various other hematopoietic progenitors have been characterized. Forexample, hematopoietic progenitor cells include those cells which arecapable of successive cycles of differentiating and proliferating toyield up to eight different mature hematopoietic cell lineages. At themost primitive or undifferentiated end of the hematopoietic spectrum,hematopoietic progenitor cells include the hematopoietic “stem cells.”These rare cells, which represent 1 in 10,000 to 1 in 100,000 of cellsin the bone marrow, each have the capacity to generate>10¹³ mature bloodcells of all lineages and are responsible for sustaining blood cellproduction over the life of an animal. They reside in the marrowprimarily in a quiescent state and may form identical daughter cellsthrough a process called self-renewal. Accordingly, such an uncommittedprogenitor can be described as being “totipotent,” i.e., both necessaryand sufficient for generating all types of mature blood cells.Progenitor cells which retain a capacity to generate all blood celllineages but which can not self-renew are termed “pluripotent.” Cellswhich can produce some but not all blood lineages and can not self-reneware termed “multipotent.”

The protein of the invention is useful to preserve any of theseprogenitor cells, including unipotent progenitor cells, pluripotentprogenitor cells, and/or totipotent progenitor cells. The protein hasdemonstrated particular efficacy in preserving hematopoietic progenitorcells, however, the protein can be used to preserve progenitor cells ofother tissues such as those mentioned above. Because of its propertiesin controlling the hematopoietic process, the protein of the inventionis also referred to herein as “pylartin” or “the pylartin protein.”However, it should be recognized that the protein is also useful in thepreservation and maintenance of progenitor cells in non-hematopoietictissues.

The pylartin protein is especially useful in preserving hematopoieticprogenitors in mammals such as humans, mice, rats, etc. In the human,primitive mature hematopoietic progenitor cells can be identified asbelonging to a class of cells defined by their expression of a cellsurface antigen designated CD34. These cells may be referred to as CD34⁺cells. In the mouse, hematopoietic progenitor cells may be referred toas Sca⁺Lin⁻ cells, reflecting their cell surface antigen signature.Other mammalian species exhibit similar signature properties identifyinghematopoietic progenitor cells. Hematopoietic progenitors can also beidentified by their expression of the flk2/flt3 receptor.

Human hematopoietic progenitor cells which express the CD34 antigenand/or the flk2 receptor are referred to herein as “primitive progenitorcells.” By contrast, hematopoietic cells which do not express either theCD34 antigen or the flk2 receptor are referred to as “mature progenitorcells.”

Generally, the pylartin protein is effective to preserve progenitorcells which express the CD34 antigen, or the flk2 receptor. Theprogenitor cells can include cells modified to express the CD34 antigenor flk2 receptors on their surface. In a preferred case, the protein hassignificant binding affinity for flk2 receptor on the cells, whereinbinding of the protein with the flk2 receptor mediates the inhibition ofdifferentiation of the cells.

The pylartin protein mediates a “preservation” of progenitor cells. Bythis is meant that the peptide either inhibits differentiation of theprogenitor cells, or induces their proliferation withoutdifferentiation. In particular, by inhibiting differentiation processes,it is to be understood that the peptide significantly lowers the rate atwhich cells differentiate, and it may in fact completely stop theseprocesses by possibly maintaining progenitor cells in a quiescent or G0state of the cell cycle. However, the protein does so without killingthe cells in significant numbers. In this sense, pylartin issignificantly distinguished from factors which inhibit or interfere withcellular processes (e.g., DNA replication, protein synthesis), and whichthereby induce significant cell mortality.

The pylartin protein of the invention can be obtained from a naturalsource, namely being derived from a legume. A legume is a fruit or seed(e.g., a pea or bean) of a leguminous plant, which are plants from afamily (Leguminosae) of dicotyledonous herbs, shrubs, and trees bearing(nitrogen-fixing bacteria) nodules on their roots. More specifically,the pylartin protein can be obtained from members of the tribePhaseoleae. In particular, the protein can be obtained from hyacinthbeans (Dolichos lab lab), kidney beans, e.g., red kidney beans(Phaseolus vulgaris), or the black-eyed pea (Vigna senensis). In itsnative form obtained from such natural sources, the protein appears tobe a mannose/glucose-specific legume lectin.

In one embodiment, the pylartin protein comprises a peptide chaindesignated β(beta), which comprises the sequence AQSLSFSFTKFD (SEQ IDNO:1). Preferably, the βpeptide comprises the sequence AQSLSFSFTKFDLD(SEQ ID NO:2). The β peptide has a molecular weight in the range ofabout 12-20 kD, typically about 13 kD.

In an alternative embodiment, the pylartin protein comprises a peptidechain designated α (alpha), which comprises the sequence VVAVEFD (SEQ IDNO:3). Preferably, the α peptide comprises the sequence TDSYVVAVEFD (SEQID NO:4). The a peptide has a molecular weight in the range of about15-20 kD, typically about 16 kD.

In another embodiment, the protein can include monomeric, dimeric andmultimeric proteins, in which one, two, or more than two peptide chains,respectively, occur together as subunits of a single proteinaceoussubstance, whether of natural or synthetic origin. The subunits of suchdimers and multimers may be the same or they may be different. A highlypreferred dimer according to the invention is a heterodimer comprisingone alpha peptide and one beta peptide (αβ).

The pylartin heterodimers (αβ) may aggregate, presumably by non-covalentinteractions, to form a tetramer (αβ)₂ with a mass of about 60 kD. Thetetramer appears to be the most common naturally occurring form of theprotein, but other multimers, e.g., concatamers of the heterodimer,i.e., (αβ)_(n) wherein n is a positive integer larger than 2, may beobservable by SDS-PAGE, in mass increments of about 25-30 kD.Accordingly, the invention encompasses all of these configurations. Inits mature form as an α₂β₂ tetramer, the pylartin protein retainsbiological activity at pH 2-10; multiple freeze-thaw cycles; at 60° C.for ten minutes; and at 4° C. for several months.

Certain molecular features of the pylartin protein indicate that theprotein is related to known plant lectins, specifically to lectinsderived from legumes. For example, the N-terminal region of the αpeptide includes a plant lectin consensus sequence, i.e., residues 5-12of SEQ ID NO:3. Also, the pylartin protein can occur as a tetramer, astructure common to many lectins.

Other features of the pylartin protein are remarkable and unexpected.For example, the protein of the invention has been obtained, asdescribed below, from the red kidney bean, which is known to produceanother lectin (i.e., phytohemagglutinin) in much larger quantities.That a plant would express two such distinctly different lectins isunexpected It is known that certain plant proteins, such as theaforementioned phytohemagglutinin, have physiological effects inmammals. Nonetheless, it is not expected that any protein ofindeterminate function from one taxonomic kingdom would also act as aspecific regulator of progenitor cells in another kingdom. Surely such aphenomenon is unexpected and unexplained. In addition, whereas certaineukaryotic proteins are identified as playing a role in developmentalcontrol (e.g., Tan-1 for hematopoietic lineages), no comparable role hasyet been defined for plant lectins.

As a result of the present invention, numerous utilities becometechnically feasible. The method of the invention can include contactingthe progenitor cells with the pylartin protein in vitro, ex vivo, or invivo. For example, the pylartin protein finds a utility, inter alia, inthat it enables ex vivo maintenance of hematopoietic progenitor cellsisolated from either normal or malignant (e.g., leukemic) bone marrow.Accordingly, the protein can be employed in the culture of mononuclearcells derived from a source of such cells, for example, from bonemarrow, umbilical cord blood, placental blood, or peripheral blood.Alternatively, the pylartin protein can be used in conjunction withgrowth factors such as colony stimulating factors (CSFs) (e.g., IL3,GM-CSF, G-CSF, M-CSF), interleukins (e.g., IL1 through IL15) and KL invitro to selectively induce proliferation and terminal differentiationof mature progenitors while leaving a highly enriched population ofprimitive progenitors. U.S. Pat. Nos. 5,472,867 and 5,186,931 describerepresentative methods of using CSFs and interleukins (ILs) to expandprogenitor cell populations in the contexts of, respectively, cancerchemotherapy and bone marrow transplants. In a preferred case accordingto the present invention, the method further includes contacting theprogenitor cells with flk2 ligand in an amount sufficient to selectivelyexpand the number of progenitor cells without inducing differentiationthereof.

The pylartin protein also enhances survival of progenitor cells whencultured in vitro. For example, a process of in vitro selection can beused which involves using the protein to preserve progenitor cells in asubstantially quiescent state in culture, while using a cytotoxic agentwhich exhibits selective toxicity for proliferating cells, e.g., to killcells passing through cell cycle (“cycling cells”). Suitable cytotoxicagents include, for example, compounds such as adriamycin,cyclophosphamide, taxol or other taxane, cisplatin, or 5-fluorouracil.The method is useful to preserve quiescent progenitor cells. The methodis effective even when the progenitor cells are substantially free ofstromal cells, cells which are considered to normally be necessary forprogenitor cell maintenance and proper hematopoietic reconstitution.Pylartin improves the ability to functionally select stem cells eitheralone or with other factors. Such functional selection methods, includethe method reported by Berardi et al. (1995) where selection is madeusing a combination of KL and IL3 with 5-FU.

Ex vivo purging protocols can be used to selectively eliminateneoplastic cells by targeting the elimination of actively cycling cells.By preserving progenitor cells in a quiescent state, the protein of theinvention preserves normal progenitor cells, while the cycling cells arekilled. Once the progenitors cells have been purged of malignant cyclingcells, they can be returned to the patient, and permitted to resumenormal proliferation and differentiation. In one especially usefulscenario, the pylartin protein allows for functional selection of normalprogenitor cells from a leukemic bone marrow. Such functional selectionand purification of primitive stem cells can also be used to enableallogeneic transplant procedures.

The pylartin protein also enables ex vivo manipulation of hematopoieticprogenitor cells for use in gene therapy by maintaining cells in liquidculture. For example, by preserving hematopoietic progenitor cells inculture for more than two weeks, the protein enables increased targetingefficiency by viral vectors that enter non-replicating cells (e.g.,vectors such as adeno-associated viruses), and provides longer periodsfor the evaluation of the resultant cell populations to determineefficiency of transfection. Thus, in another embodiment, the method canbe used in conjunction with methods of transfecting an exogenous DNAsequence into somatic cells. The method can then include transfectingprogenitor cells selectively preserved by the pylartin protein.

The invention also has utility in conjunction with therapies, e.g.,cancer therapies, which employ irradiation. Specifically, because thepylartin protein maintains progenitor cells in a quiescent state,administration of the pylartin protein to a mammalian subject in vivoallows the use of increased levels of total body irradiation toeliminate neoplastic cells, while leaving quiescent cells relativelyunaffected. The protein can be employed in conjunction with othercytoprotective substances such as IL-1 to obtain an enhanced orcomplementary effect.

Thus, the method can involve treating a mammalian subject in need ofhematopoietic therapy. For example, a tissue sample includinghematopoietic progenitor cells can be obtained from the subject. Thenthe tissue sample can be cultured ex vivo in the presence of thepylartin protein to preserve the progenitors, while allowing cyclingcells to proliferate, differentiate and die. The cultured cells becomehighly enriched in the primitive progenitor cells. Meanwhile, the mammalcan be subjected to conditions sufficient to effect myeloablation, e.g.,bone marrow irradiation, whole body irradiation, or chemically-inducedmyeloablation. Finally, the progenitor-enriched cultured cells can beadministered to the mammal following the myeloablation to reconstitutethe hematopoietic system of the mammal. While described here as anautologous procedure, the skilled practitioner will recognize that themethod can be readily adapted to transplant of progenitor-enriched cellsfrom one individual to another.

The pylartin protein also improves hematopoietic competence in a mammal,i.e., the mammal's ability to generate functional mature blood elements.For example, a tissue sample comprising hematopoietic progenitor cellscan be cultured in a growth medium containing the pylartin protein in anamount sufficient to preserve the progenitor cells and to providecultured cells enriched in the progenitor cells. Then, the enrichedcultured cells are transfused to the mammal to provide progenitor cellsfor generating blood cellular components in the mammal. This method canfurther comprise ablating hematopoietic tissues in the mammal prior tothe transfusing. In addition, the method can use a tissue samplecomprising peripheral blood, umbilical cord blood, placental blood, orbone marrow. Preferably, the tissue sample is autologous to the mammal.The tissue sample can also be at least substantially free of stromalcells.

The invention further includes a composition for maintaining viabilityof progenitor cells ex vivo or in vitro. The composition comprises aculture medium suitable for growth and maintenance of mammalian cells inculture, along with an amount of the pylartin protein sufficient topreserve progenitor cells as described herein. Virtually any cell ortissue culture medium can be modified for the preservation ofprogenitors in this way. Ready-to-use receptacles, e.g., blood bags,microtiter plates, and tissue culture flasks, and the like, can also beprovided with the pylartin protein (with or without culture medium orother active components) to promote culture of progenitor cells.

Also, the invention includes a method for preserving progenitor cells ina mammal in vivo. In this approach, the method comprises administeringto the mammal the pylartin protein in an amount sufficient to preserveprogenitor cells of the mammal in a substantially non-proliferativestate. The mammal is then exposed to myeloablative conditions sufficientto effect ablation of proliferating myeloid cells but sparingnon-proliferating progenitor cells. Following the ablative treatment,proliferation or differentiation of the preserved progenitor cells isreestablished, usually by administering to the mammal a cytokine in anamount sufficient to improve the viability of the progenitor cells.Preferred viability-improving cytokines include, for example, IL1, IL3,IL6, IL11, KL, or a combination thereof More preferably, thereestablishing comprises administering to the mammal aproliferation-stimulating amount of the flk2 ligand. According to thismethod, the pylartin protein can be used to enhance autologous bonemarrow transplantation techniques in which lethal doses of radiationand/or chemotherapy are followed by reinfusion of stored marrow.

An effective amount of pylartin protein can be administered to a mammalby any convenient approach, such as parenteral routes, e.g., intravenousinjection, or preferably by enteral routes. Oral administration routesare expected to be useful since lectins typically resist oral/gastricdegradation, and can exhibit substantial bioavailability by thisapproach. The skilled artisan recognizes the utility and limitations ofvarious methods of administration and can adjust dosage (posology)accordingly.

Other therapeutic utilities also present themselves to the skilledpractitioner as being enabled by the invention. Such other utilitiesinclude, for example, expanding progenitor cell populations ex vivo toincrease chances of engraftation, improving conditions for transportingand storing progenitor cells, and removing a fundamental barrier toenable gene therapy to treat and cure a broad range of life-threateninghematologic diseases such as sickle cell anemia and thalassemia.

The protein of the invention can also be used as a specific probe forthe identification or localization of progenitor cells. Since theprotein binds specifically to primitive progenitor cells, a compositionincluding the protein linked to a detectable moiety such as afluorescent marker can be used to specifically label and identifyprogenitor cells. Thus, cell sorting to isolate progenitor cells can beperformed, as can histologic localization of progenitor cells intissues. The skilled artisan can select the type or marker moiety to beemployed according to the task to be performed, since numerous methodsof labeling proteins are known in the art.

The following examples are provided to assist in a further understandingof the invention. The particular materials and conditions employed areintended to be further illustrative of the invention and are notlimiting upon the reasonable scope thereof.

EXAMPLE 1 Isolation of the Pylartin Protein from Kidney Beans andHyacinth Beans

Two hundred fifty grams (250 g) of kidney beans obtained from acommercial source (Stokes Seed Company, Buffalo, N.Y. or W. Atlee Burpee& Co, Warminister, Pa.) was pulverized in a coffee grinder. (This methodcan also be used to isolate material from hyacinth beans or other beanscontaining an active substance in accordance with the invention.) Thebean powder was added to 1 L of an alkaline lectin binding buffer (LBB;e.g., 50 mM Tris/HCl, pH 7.0, containing metal cations: 1 mM each ofMgCl₂ and CaCl₂) and incubated with constant mixing overnight at 4° C.The particulate matter was pelleted by centrifugation at 10,000×g for 20min and the supernatant was retained.

The protein binds specifically to mannose, enabling a single steppurification of the protein. To remove contaminants that could interferewith mannose-agarose affinity chromatography, the bean supernatant wasexposed to ammonium sulfate precipitation using 60% (0.6 g/mL). Theprecipitate was resuspended, and dialyzed overnight at 4° C. againstLBB, to provide the starting material for affinity chromatography. Thedialyzed ammonium sulfate fraction was incubated with mannose covalentlycoupled to Sepharose 4B (Sigma Chemical Co., St. Louis, Mo.) overnighton a rocker at 4° C. The gel was then washed extensively with LBB andpelleted by gravity sedimentation. Lectins were eluted with 250 mMmethyl α-D-methylpyranoside (Sigma Chemical Co., St. Louis, Mo.) in LBBfor two days on a rocker at 4° C., pelleted by density sedimentation,and the lectin-containing supernatant aspirated. The supernatantcontaining the pylartin protein was dialyzed overnight against LBB toremove sugar, and the resulting dialysate was sterile filtered, andstored at −20° C.

FIG. 1 shows SDS-PAGE analysis of purified pylartin protein isolatedfrom hyacinth beans, and purified by mannose-agarose. Pylartin samplesand molecular markers (Integrated Separation Systems, Natwick, Mass.)were analyzed under reducing conditions on a 10-27% gradientpolyacrylamide gels (Integrated Separation Systems, Natwick, Mass.) andvisualized by Coomassie Brilliant Blue staining (Sigma Chemical Co., St.Louis, Mo.). Molecular weights are indicated on left side of the gel;alpha and beta subunits are indicated for each on the right side of eachgel and numbers indicate isoforms of subunits. Four isoforms of thealpha subunit and one of the beta subunit of pylartin were detected inthe hyacinth bean.

EXAMPLE 2 flk2 Receptor 3T3 Assay Specifically Quantitates BiologicalActivity of the Pylartin Protein

The pylartin protein interacts with the mammalian flk2 tyrosine kinasereceptor. A specific and quantitative biological assay using NIH 3T3fibroblasts transfected either with a chimeric receptor consisting ofthe extracellular portion of the murine flk2 receptor combined with theintracellular portion of the human Fms receptor (Lemischka et al.,unpublished) or with the full length human receptor (Small et al. 1994)can be used to evaluate lectin biological activity during purification.Serial two-fold dilutions of lectin samples across rows of a 96 wellplate allowed for greater than a thousand-fold range to access flk2 3T3biological activity. The assay requires either the murine or human flk2/flt3 ligand (FL) (Lyman et al. 1993; Hannum et al. 1994) or thepylartin protein to rescue flk2-transfected cells from death.

Specifically, remove 3T3 cells from stock culture plates by washingcells cultured in tissue culture plates (Becton Dickinson Labware,Lincoln Park, N.J.) twice in Hank's buffered saline (HBSS; GibcoLaboratories, Grand Island, N.Y.) followed by addition of non-enzymaticcell dissociation buffer (Gibco) for 15 minutes at room temperature, andsubsequently wash cells in medium. Culture flk2 3T3 cells at a finalconcentration of 3,000 cells per well in a volume of 100 μL ofserum-defined medium containing 10% AMV (Gibco) and 90% Dulbecco'smodification of Eagle's medium (DMEM; Gibco) in 96 well plates. Underthese assay conditions, cells die after two to four days of culture in ahumidified incubator at 37° C. and 5% CO₂ unless exogenously addedligand rescues cells from death. Each 96 well plate contains calf serum,which stimulates all 3T3 cells, as a positive control and medium only asa negative control. Full-length Fms-transfected 3T3 cells (biologicalresponse shown in Tessler et al. 1994) serve as receptor-transfectedcontrol target cells, and parent 3T3 cells serve as untransfectedcontrol cells. Proliferation and cell survival is quantitated byaddition of XTT (Diagnostic Chemicals Ltd, Charlottetown, Prince EdwardIsland, Canada), a tetraformazan salt cleaved by actively respiringcells (Mosmann 1983), and quantitated spectrophotometrically using aVmax kinetic plate reader (Molecular Devices Corp., Mountain View,Calif.), and recorded as either relative activity (units/mL) or asspecific activity (units/mg). One unit of biological activity is definedas the reciprocal dilution at which half-maximal stimulation of cells isdetected. FIGS. 2A-2D show that pylartin isolated from red kidney beansspecifically stimulates flk2 3T3 cells in an IL1-dependent manner.Pylartin biological was tested over a 1,000-fold range on flk2 /Fms 3T3cells (FIGS. 2A and 2B) and parent untransfected 3T3 cells (FIGS. 2C and2D) in the absence (FIGS. 2A and 2C) and presence (FIGS. 2B and 2D) of10 ng/mL human recombinant interleukin 1-alpha (IL1-α) (BioSourceInternational, Camarillo, Calif.) and quantitated by XTT. The pylartinprotein stimulated proliferation of flk2 /Fms 3T3 cells only in thepresence of IL1, and did not stimulate proliferation of untransfected3T3 cells regardless of whether IL1 was present or not.

Examples 3-10, below, show that the protein of the invention preservesprogenitors cells in vitro. Pylartin isolated from red kidney beans andhyacinth beans specifically preserves progenitors derived from murinefetal liver cells and human cord blood cells in liquid culture.

EXAMPLE 3 The Pylartin Protein Preserves Human and Murine HematopoieticProgenitor Cells In Vitro

Hematopoietic progenitor cells range in frequency from about 1:10,000 toabout 1:100,000 of mononuclear cells in human bone marrow, umbilicalcord blood, and under certain conditions, peripheral blood. Enrichmentof progenitor cells for in vitro culture involves isolating mononuclearcells from a sample of whole blood or blood marrow by densityseparation. Specifically, the sample is gently layered over Ficoll-PaquePlus (Pharmacia Biotech, Piscataway, N.J.) in a 50 mL conical tube(Becton Dickinson, Franklin Lakes, N.J.) and the samples are processedaccording to the manufacturer's directions.

Enrichment of stem cells greater than a hundred-fold from mononuclearcell preparations can be achieved using antibody-labeled magnetic beadtechnology. Two commercial methods (DYNABEADS, available from DynalCorp., Lake Success, N.Y., and MINIMACS multiparameter sorting, MiltenyiBiotec, Auburn, Calif.), rely on positive selection of CD34⁺ cells.Another method (STEMSEP CD34³⁰ /CD38⁻, available from Stem CellTechnologies, Inc., Vancouver, BC) isolates stem cells by negativeselection (glycophorin A, CD14, CD3, CD16, CD2, CD66b, CD56, CD36, CD24,CD45RA, CD19, CD38), resulting in cells with the surface phenotype ofCD34⁺, CD36⁻, CD38⁻, CD45RA⁻.

The pylartin protein is used to preserve the isolated progenitor cells,either within a population of mononuclear cells or in enriched CD34⁺cells, in liquid culture containing serum-defined medium (AIMV-V fromGibco, Grand Island, N.Y.; BIT9500 from Stem Cell Technologies,Vancouver, BC; or X-VIVO 10 from BioWhittaker, Walkersville, Md.) in anincubator maintained at 37° C. and flushed with 5% CO₂, for two to fourweeks. The resultant cell population is characterized by flow cytometry,and hematopoietic colony assays are performed to enumerate and evaluatethe functional status of the progenitor cells.

Human hematopoietic stem cells express the cell surface antigenphenotypic profile of CD34⁺⁺ CD38⁻ Thy⁺, but do not express antigens formature blood cell lineages (CD3, CD19, etc.) (Peltzer et al. 1996).Specifically, cells are incubated with fluorescent-labeled antibodies(e.g., Coulter Corp., Hialeah, Fla.) following manufacturer's directionsand are evaluated for marker expression by flow cytometry (e.g., EpicsElite, Coulter Corp.).

Hematopoietic colony assays indirectly assess the frequency andprimitive status of progenitors in a population of cells. Cells arecultured in semi-solid methylcellulose containing growth factors foroptimal stimulation of cell proliferation and differentiation (Stem CellTechnologies, Vancouver, BC).

EXAMPLE 4 Pylartin Preserves Murine Fetal Liver CD34⁺ Cells in Culture

FIG. 3 shows that purified pylartin isolated from red kidney beans(Pylartin-RK) and hyacinth beans (Pylartin-H), either alone or togetherwith increasing concentrations of a cytokine cocktail (mIL1, mIL3, mKL),preserves murine fetal liver CD34⁺ cells for six days in liquid culturemedium containing 10% calf serum in DMEM. After fifteen days thecultures were harvested and cells replated in a methylcellulose colonyassay to assess frequency of functional progenitors. NT indicates nottested. Mononuclear cells cultured in the presence or absence ofpylartin with either no exogenous cytokines or a cocktail ofearly-acting recombinant murine cytokines (mIL1, mIL3, mKL) (BioSourceInternational, Camarillo, Calif.) all at either 5 ng/mL or 50 ng/mL.Flow cytometric analysis of remaining viable cells shows that theproportion of CD34⁺ cells in culture wells containing pylartin increasesin a dose-dependent manner of early acting cytokine cocktail is presentbut not in medium control.

EXAMPLE 5 Pylartin Preserves Murine Hematopoietic Progenitors in Culture

The pylartin protein, either alone or in combination with either IL1 orFL, preserves functional hematopoietic progenitors for at least twoweeks in culture. Specifically, FIG. 4 shows the preservative effect ofpurified pylartin on murine hematopoietic progenitors cultured fifteendays in liquid culture medium containing 10% calf serum in DMEM.Evaluation of functional progenitors was tested by harvested cells inliquid culture and replated in methylcellulose colony assays (Stem CellTechnologies, Vancouver, British Columbia) according to themanufacturer's instructions. Colonies derived from individualprogenitors were scored after two weeks; results were normalized forfrequency of colonies per 100,000 cells initial seeded. Each of thepylartin samples preserved progenitors in the absence of the exogenouscytokine cocktail (left bars each group); variable differences wereobserved for cytokine cocktail-containing pylartin samples.

EXAMPLE 6 Flow Cytometric Analysis of Cell Surface Markers on CellsCultured with Pylartin Protein

The type of cells generated in methylcellulose colony assays in culturescontaining 50 ng/mL of the cytokine cocktail (FIG. 4) was determined byflow cytometry. These data are summarized in FIG. 5. Sca⁺ cells weregreatly increased in pylartin-containing cultures; other cell surfacemarkers were not particularly affected. The Sca antigen is expressed onmurine stem cells and other mature cells including T cells. In thisexperiment, T cells (CD3) were not increased. FIG. 5 shows a flowcytometric analysis of the cell surface phenotype of cells harvestedfrom cells cultured in 50 ng/mL of early-acting recombinant murinecytokines (mIL1, mIL3, mKL) (BioSource International, Camarillo,Calif.), in the presence and absence of pylartin samples, prior tocolony assay (far right bars from FIG. 4). Pylartin-RK generated14.5-fold more Sca-1 cells than medium control. Sca-1, or Ly6, is anantigen associated with primitive murine progenitors and also matureblood cells including T cells, etc. (Spangrude et al. 1991). Nodifferences were observed in the numbers of T cells (CD3), dendriticcells (CD11b), or cells expressing the Fc-gamma receptor (e.g.,granulocytes and monocytes), CD38, or the transferrin receptor (CD71)(antibodies obtained from Pharmigen, Calif.).

EXAMPLE 7 The Pylartin Protein Preserves Human CD34⁺ Cells in Culture

Pylartin also preserves human primitive progenitors. FIGS. 6A and 6Bshow that pylartin, either alone or in combination with early-actinghuman cytokines (rhIL1-a, rhIL3, rhFL, rhKL), specifically preservesCD34⁺ human cord blood cells for up to two weeks. The purified pylartinisolated from hyacinth beans (Pylartin-H) specifically preserves humancord blood cells that express the CD34 antigen for seventeen days (FIG.6A) and six days FIG. 6B) in liquid culture containing a serum-definedmedium (BIT9500, Stem Cell Technologies, Vancouver, British Columbia).Pylartin is effective to preserve CD34⁺ cells either with or withoutadded exogenous cytokines.

EXAMPLE 8 The Pylartin Protein Preserves Human Progenitors in LiquidCulture

The pylartin protein, either alone or in combination with either IL1 orFL, preserves functional progenitors for at least two weeks in liquidculture, FIG. 7 shows in this experiment the combination of pylartin andFL actually increases the number of progenitors in liquid culture underselected assay conditions. The number of colonies derived fromfunctional progenitors is several times greater where pylartin ispresent (left panel) than in medium control (right panel), with orwithout exogenous IL1 or FL.

The nature of hematopoietic progenitors preserved by pylartin in liquidculture changes over time. Cord blood mononuclear cells (mnc) werecultured in X-VIVO 10 at a concentration of 800,000 cells in 4 mL for 6,15, and 21 days with either pylartin, FL, pylartin+FL, or mediumcontrol. Functional progenitors were assessed by replating cells inmethylcellulose colony assays and scoring colonies after an additional16 days. Four types of colonies were recorded: “Blast” refers to small,dispersed colonies consisting of undifferentiated cells which mayrepresent very primitive stem cells; “Mix” refers to colonies consistingof differentiated myeloid and erythroid cells formed by multilineageprogenitors; “Myeloid” refers to colonies consisting exclusively ofdifferentiated myeloid cells; “Erythroid” refers to colonies consistingexclusively of differentiated erythroblasts or normoblasts formed byprimitive progenitors called “BFU-E” (burst-forming unit erythroid).

Colonies formed in all liquid culture wells after six days of culture(FIG. 8A). The solid line in FIG. 8A indicates too many colonies toaccurately score, which in this experiment was greater than 1,500colonies per 800,000 mnc.

After 15 days of culture pylartin but not FL preserves relatively matureprogenitors of myeloid or erythroid lineages (FIG. 8B). “Blast”-likecells and myeloid-committed progenitors appear in methylcellulose colonyassay plates from cord blood mononuclear cells preserved in liquidculture for 21 days (FIG. 8C). The relatively high frequencies ofprogenitors cultured for 15 and 21 days indicate not only preservationof progenitors but also an increase (self-renewal) of primitiveprogenitors.

EXAMPLE 9 Pylartin Preserves Progenitor Cells in Culture in aDose-Responsive Manner

Pylartin acts in a dose-responsive manner to preserve human cord bloodprogenitors. FIGS. 9A-9B show results from culturing cord bloodmononuclear samples from two donors (FIGS. 9A and 9B, respectively)using the method described in Example 8. In this case, cultures weremaintained for 12 days over a 100-fold concentration range of pylartin.Although the response differed between the two donors—both incomposition of colony types and progenitor frequency—the specificactivity of DLL, at 20,000 units/mg, is approximately the same in eachcase. TMTC indicates too many colonies to count.

EXAMPLE 10 Pylartin Preserves Progenitors in Culture by PreventingDifferentiation into Mature Blood

FIG. 10 shows that pylartin, obtained in this case from hyacinth beans,prevents IL3-induced production of cells expressing the Fc-gammareceptor after six days in liquid culture. IL3-containing culturescontained approximately 80% CD32⁺ cells, while pylartin-containingcultures contained <10% CD32⁺ cells. Cells that express FcγII and FCγIIreceptors in mice include natural killer cells, monocytes, macrophages,granulocytes, mast cells, as well as fetal thymocytes (Unkeless 1979).

The data presented in the above Examples demonstrate that pylartinpreserves primitive hematopoietic progenitors, either alone or incombination with other cytokines, and prevents IL3-induceddifferentiation in liquid culture. Indeed, the protein by itself is moreeffective in preserving progenitors than FL even when the latter issupplemented with stromal cells. While no data have yet been obtained totest whether the peptide and stromal cells are cultured together,applicants believe that a potentiation of the preservative effect of thepeptide would be maintained. Nonetheless, it is apparent that theprotein of the invention is effective in preserving primitivehematopoietic progenitors in cell culture. This implies that the proteinis effective for use in clinical methods involving explantation ofprogenitors, culture and preservation of the progenitors ex vivo, andsubsequent reimplantation (or transplantation) of the preservedprogenitors.

Thus, while there have been described what are presently believed to bethe preferred embodiments of the present invention, those skilled in theart will realize that other and further embodiments can be made withoutdeparting from the spirit of the invention, and it is intended toinclude all such further modifications and changes as come within thetrue scope of the claims set forth herein.

BIBLIOGRAPHY

The following publications, mentioned in the foregoing specification,are incorporated herein by reference for all that they disclose:

Baronedes, SH, “Bifunctional properties of lectins: Lectins redefined,”Trends Biochem. Sci. 13:480-482 (1988).

Berardi, A C et al., “Functional isolation and characterization of humanhematopoietic stem cells,” Science, 267:104-108 (1995).

Dwek, R A, “Glycobiology: More function for oligosaccharides,” Science269:1234-1235 (1995).

Gabius, H-J, “Non-carbohydrate binding partners/domains of animallectins,” Int. J. Biochem. 26:469 (1994a).

Gabius, H-J, “Lectinology meets mythology: Oncological future for themistletoe lectin?,” Trends in Glycosci and Glycotech 6:229 (1994 b).

Gowda, L R, H S Savithri, and D R Rao, “The complete primary structureof a unique mannose/glucose-specific lectin from field bean (Dolichoslab lab),” J. Biol. Chem. 269:18789-18793 (1994).

Hannum, C O et al., “Ligand for Flt3/fk2 receptor tyrosine kinaseregulates growth of hematopoietic stem cells and is encoded by variantRNAs,” Nature 368:643 (1994).

Lyman, S D et al., “Molecular cloning of a ligand for the Flt3/flk2tyrosine kinase receptor: a proliferative assay for primitivehematopoietic cells,” Cell 75:1157 (1993).

Mosmann, T, “Rapid colorimetric assay for cellular growth and survival:Application to proliferation and cytotoxicity assays,” J. Immunol.Methods 65:55-63 (1983).

Ogawa, M, “Differentiation and proliferation of hematopoietic stemcells,” Blood 87:2855 (1993).

Peltzer, A L et al., “Self-renewal of primitive human hematopoieticcells (long-term-culture-initiating cells) in vitro and their expansionin defined medium,” Proc. Natl. Acad. Sci. (USA), 93:1470-1474 (1996).

Shah, A J et al., “flt3 Ligand induces proliferation of quiescent humanbone marrow CD34⁺CD38⁻ cells and maintains progenitor cells in vitro,”Blood, 87:3563-3570 (1996). Sharon, N, and H Lis, “Lectins as cellrecognition molecules,” Science, 246:227-234 (1989).

Small, D, M Levenstein, E Kim, C Carow, S Amim, P Rockwell, L Witte, CBurrow, M Ratajczak, A M Gewirtz, and C Civin, “STK-1, the human homologof flk-2/flt3, is selectively expressed in CD34⁺ human bone marrow cellsand is involved in the proliferation of early progenitor/stem cells,”Proc. Natl. Acad Sci. USA, 91:459-463 (1994).

Spangrude, G J, et al., “Purification and characterization of mousehematopoietic stem cells,” Science 241:58-62 (1991).

Tessler, S, P Rockwell, D Hicklin, T Cohen, B-Z Levi, L Witte I RLemischka, and G Neufeld, “Heparin modulates the interaction of VEGF165with soluble and cell associated flk-1 receptors,” J. Biol. Chem.269:12456-12461 (1994).

Turhan A G, et al., “Clonal hematopoiesis demonstrated by x-linked DNApolymorphisms after allogeneic bone marrow transplantation,” N. Engl. J.Medicine, 320:1655-1661 (1989).

Unkeless, J C, “Characterization of a monclonal antibody directedagainst mouse macrophage and lymphocyte Fc receptors,” J. Exp. Med.150:580-596 (1979).

Young, J C, A Varma, D DiGiusto, and M Backer, “Retention of quiescenthematopoietic cells with high proliferative potential during ex vivostem cell culture,” Blood 87:545-556 (1996).

Zipori, D, “The renewal and differentiation of hemopoietic stem cells,”FASEB J. 6:2691-2697 (1992).

What is claimed is:
 1. A method for preserving progenitor cells,comprising contacting progenitor cells with a protein having an aminoacid sequence comprising SEQ ID NO:1 and a molecular weight of about12-20 kD, or having an amino acid sequence comprising SEQ ID NO:3 and amolecular weight of about 15-20 kD, in an amount sufficient to preservethe progenitor cells.
 2. A method according to claim 1, wherein theprotein has an amino acid sequence comprising SEQ ID NO:2.
 3. A methodaccording to claim 1, wherein the protein has an amino acid sequencecomprising SEQ ID NO:4.
 4. A method according to claim 1, wherein theprotein is obtained from a legume from the tribe Phaseoleae.
 5. A methodaccording to claim 4, wherein the protein is obtained from kidney beans,hyacinth beans, or black-eyed peas.
 6. A method according to claim 1,wherein the progenitor cells comprise hematopoietic progenitor cells. 7.A method according to claim 6, wherein the progenitor cells comprisehuman cells which express the CD34 antigen and/or the flk2 receptor. 8.A method according to claim 6, wherein the progenitor cells comprisemurine cells which express the Sca antigen but which do not expressmature blood cell lineage antigens.
 9. A method according to claim 1,wherein the method comprises contacting the progenitor cells with theprotein in vitro, ex vivo, or in vivo.
 10. A method according to claim1, wherein the method further comprises contacting the progenitor cellswith flk2 ligand in an amount sufficient to selectively expand thenumber of progenitor cells without inducing differentiation thereof. 11.A method of treating a mammal in need of hematopoietic therapy,comprising: a) obtaining a tissue sample from the mammal, the tissuesample comprising hematopoietic progenitor cells; b) culturing theprogenitor cells in the presence of a protein which preserves theprogenitor cells to provide cultured cells enriched in the progenitorcells, wherein the protein has an amino acid sequence comprising SEQ IDNO:1 and a molecular weight of about 12-20 kD, or has an amino acidsequence comprising SEQ ID NO:3 and a molecular weight of about 15-20kD; c) subjecting the mammal to conditions sufficient to effectmyeloablation; and d) administering the cultured cells to the mammalfollowing the myeloablation to reconstitute the hematopoietic system ofthe mammal.
 12. A method according to claim 11, wherein themyeloablation conditions comprise bone marrow irradiation, whole bodyirradiation, or chemically-induced myeloablation.
 13. A method ofenriching progenitor cells, comprising culturing progenitor cells in aprogenitor-preserving amount of a protein having an amino acid sequencecomprising SEQ ID NO:3 and a molecular weight of about 15-20 kD, orhaving an amino acid sequence comprising SEQ ID NO:1 and a molecularweight of about 12-20 kD, wherein the protein specifically preserves theprogenitor cells, and wherein the culturing is performed underconditions permitting preservation of progenitor cells while permittingthe number of differentiated cells to decrease.
 14. A method accordingto claim 13, wherein the progenitor cells include primitive progenitorcells.
 15. A method according to claim 13, wherein the progenitor cellsinclude mature progenitor cells.
 16. A method according to claim 13,wherein the progenitor cells are at least substantially free of stromalcells.
 17. A method according to claim 13, wherein said culturingconditions include culturing in a medium containing a cytotoxic agentwhich exhibits selective toxicity for proliferating cells.
 18. A methodaccording to claim 17, wherein the cytotoxic agent is adriamycin,cyclophosphamide, taxol or other taxane, cisplatin, or 5-fluorouracil.19. A method of improving hematopoietic competence in a mammal,comprising: a) culturing a tissue sample comprising hematopoieticprogenitor cells in a growth medium containing a protein having an aminoacid sequence comprising SEQ ID NO:1 and a molecular weight of about12-20 kD, or having an amino acid sequence comprising SEQ ID NO:3 and amolecular weight of about 15-20 kD, in an amount sufficient to preservethe progenitor cells and to provide cultured cells enriched in theprogenitor cells; and b) transfusing the enriched cultured cells to themammal to provide progenitor cells for generating blood cellularcomponents in the mammal.
 20. A method according to claim 19, whereinthe tissue sample comprises peripheral blood, umbilical cord blood,placental blood, or bone marrow.
 21. A method according to claim 20,wherein the tissue sample is autologous to the mammal.
 22. A methodaccording to claim 19, wherein said tissue sample is at leastsubstantially free of stromal cells.
 23. A method according to claim 19,further comprising ablating hematopoietic tissues in the mammal prior tothe transfusing.
 24. In a method of transfecting an exogenous DNAsequence into somatic cells, the improvement comprising transfectingprogenitor cells selectively preserved by a protein having an amino acidsequence comprising SEQ ID NO:1 and a molecular weight of about 12-20kD, or having an amino acid sequence comprising SEQ ID NO:3 and amolecular weight of about 15-20 kD.
 25. A composition for maintainingviability of progenitor cells ex vivo, comprising a cell growth mediumand a protein which preserves progenitor cells, wherein the protein hasan amino acid sequence comprising SEQ ID NO:1 and a molecular weight ofabout 12-20 kD, or has an amino acid sequence comprising SEQ ID NO:3 anda molecular weight of about 15-20 kD.
 26. A method for preservingprogenitor cells in a mammal, comprising: a) administering to saidmammal a protein which specifically preserves progenitor cells, saidprotein having an amino acid sequence comprising SEQ ID NO:1 and amolecular weight of about 12-20 kD, or having an amino acid sequencecomprising SEQ ID NO:3 and a molecular weight of about 15-20 kD, in anamount sufficient to preserve progenitor cells of said mammal in asubstantially non-proliferative state; b) exposing the mammal tomyeloablative conditions sufficient to effect ablation of proliferatingmyeloid cells but sparing non-proliferating progenitor cells; and c)following said exposing, reestablishing proliferation or differentiationof the preserved progenitor cells.
 27. A method according to claim 26,wherein said reestablishing comprises administering to said mammal acytokine in an amount sufficient to improve the viability of theprogenitor cells.
 28. A method according to claim 27, wherein saidviability-improving cytokine is IL-1, IL-3, IL-6, IL-11, KL, or acombination thereof.
 29. A method according to claim 26, wherein saidreestablishing comprises administering to said mammal aproliferation-stimulating amount of the flk2 ligand.